Establishment of a Highly Efficient In Vitro Regeneration System for Rhododendron aureum

Rhododendron aureum is identified as a vulnerable species in China. The establishment of an in vitro regeneration system assists significantly in species protection. Here, an in vitro regeneration system was developed using both direct and indirect organogenesis pathways. The role of thidiazuron (TDZ) in different developmental stages was also investigated. The leaves of wild-harvested R. aureum plants were used for callus induction after hydroponic cultivation. The optimal formula was found to be woody plant basal medium (WPM) supplemented with 0.5 mg & BULL;L-1 TDZ and 0.5 mg & BULL;L-1 3-indolebutyric acid (IBA), while the optimal formula for the subculture and induction of adventitious buds was WPM containing 0.1 mg & BULL;L-1 TDZ and 0.5 mg & BULL;L-1 IBA. The leaves from tissue-cultured seedlings were then used for the induction of bud clusters. An association was observed between the differentiation of bud clusters and the ratio of auxin and cytokinin. The optimal formula for the induction of bud clusters was WPM containing 0.5 mg & BULL;L-1 TDZ and 0.1 mg & BULL;L-1 IBA, yielding a 50% induction rate and the maximum number of buds. Higher concentrations of TDZ were found to be beneficial for bud proliferation, while a lower concentration was conducive to stem elongation. The optimal formula for subculture was WPM containing 0.1 mg & BULL;L-1 TDZ and 0.5 mg & BULL;L-1 IBA, while that for stem elongation was WPM supplemented with 0.002 mg & BULL;L-1 TDZ and 0.5 mg & BULL;L-1 IBA. Only IBA was found to be necessary for rooting, with increased IBA concentrations leading to an increased number of roots and earlier root formation, with larger callus areas; thus, an IBA concentration of 1.0 mg & BULL;L-1 was found to be optimal for the rooting of R. aureum. After hardening the seedlings for two days, a substrate composed of vermiculite and peat soil in a 10:1 ratio was identified as a transplantation formula. This system provides directions both for the protection of endangered species and the promotion of industrial development.